2.6. Apoptosis analysis
Apoptotic cells were measured using an Annexin V-FITC Apoptosis Detection kit
(BD Biosciences, USA) according to the manufacturer’s protocol. Briefly, treated cells
were washed in cold 1% sodium azide in PBS and then the cell pellets were resuspended
in 1 binding buffer at 1 106 cells/ml. A sample (100 ll) of the solution
was transferred to a culture tube and double-stained with 5 ll of annexin V-FITC
and 5 ll of PI for 15 min at RT in the dark. 400 ll of 1 binding buffer was added
to the mixture before analysis using FACScan flow cytometry equipped with Cell-
Quest software. A total of 20,000 cells were analysed for each sample.